Electrochemical detection of antibiotic drug rifampicin

1. Electrochemical Estimation of Anti-Tuberculosis Drug on
GO@CuO-Nanocomposite Modified GCE
Presented by:
Dr. Pinky Sagar
TFB-Fellow
Physics-MMV, BHU, Varanasi-221005
International Conference on Recent Trends in Physics
cum Alumni Meet-2024
Department of Physics, Isc., BHU
1

2. 2
 What is anti-biotic resistance?
 Why anti-biotic resistance is concern?
 Rifampicin and its side effects
 Electrochemical estimation of Rifampicin
 Assessment of sensor parameters
 Conclusion
 Acknowledgements
Objectives

3. 3
Antibiotic Resistance

4. How Antibiotic Resistance Spreads
4
Animals get antibiotics
and develop resistant
bacteria in their
systems
John gets antibiotics
and develops resistant
bacteria in his system
John stays at home
and in the general
community, spreads
resistant bacteria.
John gets care at
hospital, nursing home
or other inpatient care
facility.
Resistant germs spread
directly to other patients
or indirectly on unclean
hands of healthcare
providers.
Patients
go home.
Resitant bacteria spread to
other patients from surfaces
within the healthcare facility.
Drug-resistant bacteria in the animal feces can
remain on crops and be eaten. These bacteria
can remain in the human digestive system.
Drug-resistant bacteria
can remain on meat. When
not handled or cooked
properly, the bacteria can
spread to humans.
Fertilizer or water containing
animal feces and drug-resistant
bacteria is used on food crops.

5. 5
Impacts of Anti-biotic Resistance
• ↑ Risk of spreading infections
• Makes Infections harder to treat, prolonged illness
• ↑ Healthcare costs
• Identified antibiotic resistance as one of the top
10 threats to global health
• Launched GLASS (global Antimicrobial Resistance
and Use Surveillance System) in 2015
Recognition by WHO
• Surveillance of AMR in microbes causing TB,
Vector Borne diseases, AIDs, etc.
• National Action plan on AMR (2017) with one
health approach
• Antibiotic Stewardship Program by ICMR
India’s Initiatives against AMR
• Carbapenem antibiotics stop responding due to
antimicrobial resistance (AMR) in K. pneumonia
• AMR Mycobacterium tuberculosis causing
Rifampicin-Resistant TB (RR-TB)
• Drug-reistant HIV (HIVDR) making antiretroviral
(ARV) drugs ineffective.
Examples

6. 6
• Tuberculosis
• Bacterial Infection
• Meningococcal disease
Uses:
Excess dose of Rifampicin
Liver Complications Jaundice
Hypersensitivity

7. 7
GO
Sonication
2h
GO Suspension
CuO
CuO+GO Suspension
Probe
Sonication
6h
Dried at 80 oC
12 h
Calcination at 350 oC
4 h
GO@CuO
GO@CuO GCE
GO@CuO Synthesis

8. 8
(a) (c)
(b)
Figure: TEM images of (a) GO (b) CuO (c) GO@CuO Composite.
Characterizations of GO@CuO

9. GO@CuO C
O Cu
9
(a) (b)
Figure: (a) SEM image and (b) EDX color mapping of GO@CuO Composite.
SEM of GO@CuO

10. 500 1000 1500 2000 2500 3000
Intensity
(arb.
unit)
Wavenumber (cm-1
)
GO@CuO
D
G
10
Figure: (a) Raman spectrum of GO@CuO. (b) FTIR spectra of GO@CuO.
200 400 600
Wavenumber (cm-1
)
Ag
2Bg
Si/SiO2
(a) (b)
Raman and FTIR Spectroscopy
500 1000 1500 2000 2500 3000 3500 4000
Transmittance
(a.u.)
Wavenumber (cm-1
)
CuO
Epoxide
C-O-H
C-O
C=O
-OH
-CH
Stretching

11. 11
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
1
2
3
4
5
6
7
8
Current
(mA)
Potential (V) vs. Ag/AgCl
Mod. GCE
Mod. GCE + RFP
Bare GCE + RFP
Bare GCE
(a)
0.30 0.35 0.40 0.45 0.50 0.55 0.60
2
3
4
5
6
7
8
Current
(mA)
Potential (V) vs. Ag/AgCl
43.53 µM
0 µM
(b)
0 7 14 21 28 35 42
4
5
6
7
8
Current
(mA)
Concentration (mM)
Y = (0.10±0.003) X + (4.21±0.05)
R2
= 0.99
(c)
Figure: (a) DPV of bare GCE in absence and presence of RFP, Mod. GCE in absence and presence of RFP in 0.1 M PBS (pH =
7.4) (b) DPV response of Mod. GCE upon addition of various concentration of RFP (c) corresponding calibration plot in 0.1 M
PBS (pH = 7.4).
Note: The therapeutic range for rifampicin in the blood is generally considered to be between 8 to 24 µM for the treatment of
tuberculosis.
Sensing of Rifampicin

12. 12
0.2 0.4 0.6 0.8
-10
-8
-6
-4
-2
0
2
4
6
Current
(mA)
Potential (V) vs. Ag/AgCl
Bare GCE
Bare GCE + RFP
Mod. GCE
Mod. GCE + RFP
(a)
0.2 0.4 0.6 0.8
-12
-10
-8
-6
-4
-2
0
2
4
6
Current
(mA)
Potential (V) vs. Ag/AgCl
(b)
0 10 20 30 40 50
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Current
(mA)
Concentration (mM)
Y = (0.07±0.001) X + (2.74±0.02)
R2
= 0.99
(c)
0 10 20 30 40 50
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Y = (0.08±0.004) X + (2.70±0.06)
R2
= 0.98
Current
(mA)
Concentration (mM)
(d)
Figure: (a) CV of bare
GCE, bare GCE+RFP, Mod.
GCE and Mod. GCE+RFP.
(b) CV response of Mod.
GCE upon addition of
various concentration of
RFP (c) corresponding
calibration plot for
oxidation current and (d)
reduction current.

13. 13
0.2 0.4 0.6 0.8
-12
-10
-8
-6
-4
-2
0
2
4
6
Current
(mA)
Potential (V) vs. Ag/AgCl
0 10 20 30 40 50
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Current
(mA)
Concentration (mM)
Y = (0.10±0.003) X + (2.74±0.04)
R2
= 0.99
0 10 20 30 40 50
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Current
(mA)
Concentration (mM)
Y = (0.09±0.003) X + (2.98±0.05)
R2
= 0.99
(c) (d) (e)
0 10 20 30 40
4
5
6
7
8
Current
(mA)
Concentration (mM)
Y = (0.13±0.03) X + (3.9±0.04)
R2
= 0.99
(a) (b)
0.30 0.35 0.40 0.45 0.50 0.55 0.60
2
3
4
5
6
7
8
Current
(mA)
Potential (V) vs. Ag/AgCl
0 µM
43.53 µM
Figure: (a) DPV
response of Mod. GCE
upon addition of various
concentration of RFP
(R-CIN) (b)
corresponding
calibration plot (c) CV
response of Mod. GCE
upon addition of various
concentration of RFP
(R-CIN) (d)
corresponding
calibration plot for
oxidation and (e)
reduction current
Testing in Pharmaceutical Formulation

14. 14
Amount of RFP from
capsule (µM)
Amount of standard
RFP drug spiked (µM)
Total RFP in
sample (µM)
Total RFP found
(µM)
Percentage recovery
6
4 10 9.69 96.96
5 11 11.32 102.90
7 13 13.21 101.61
8 14 14.32 102.28
Recovery Test

15. 15
Comparison Data
Electrode Method Linear
Range (µM)
LOD(µM) Matrix Ref.
C-Dots@Cu2FeO4/CPE SWV 0.07 – 8.0 0.022 B-R Buffer (pH=7) Shiri et. al, 2020
MWCNTs-CoTHPP/GC LSV 0.01 – 5000 0.008 Acetate Buffer (pH=4.7) Sonkar et. al, 2020
PMel-Aunano/GCE CV 0.08 – 15 0.03 PBS (pH=7) Srivastava et. al,
2021
MWCNTs−Mo2C/GCE CV 0.5 – 74 0.045 PBS (pH=7) Huang et. al, 2022
MWNT/GCE DPV 0.04 – 10 0.0075 Acetate Buffer (pH=3.5) Yan et. al, 2022
SWV 0.04 – 10 0.0113
Co/Fe3O4NPs/MWCNTs/GCE CV 2 – 20 0.032, 0.413 PBS (pH=7.5) Chokkareddy et al,
2023
GO@CuO/GCE
DPV 0.05 – 35 0.005 PBS (pH=7.4) Present Work
CV(Oxi.) 0.05 – 35 0.006
CV(Red.) 0.05 – 29 0.008
GO@CuO/GCE
DPV 0.05 – 25.5 0.006 Pharmaceutical
formulation (pH=7.4)
Present Work
CV(Oxi.) 0.05 – 35 0.007
CV(Red.) 0.05 – 29 0.011

16. 16
Mechanism

17. 17
Conclusion
• GO and then GO@CuO were successfully synthesized and
characterized.
• Significantly lowers the oxidation potential of Rifampicin,
evidenced by both DPV and CV.
• LOD and sensitivity are 5 nM, 1.42 µA µM-1cm-2, 11 nM, 1.86 µA
µM-1cm-2 for standard and pharmaceutical drug, respectively.
• DPV assessment was validated by CV also.
• Through catalytic activity, facilitated charge transfer, and
modification of surface properties, GO@CuO nanomaterials can
effectively enhance the efficiency and sensitivity of electrochemical
sensors and devices.
Conclusion and Remarks

19. 19
0 4 8 12 16 20 24 28 32
0
20
40
60
80
100
120
Days
Change
in
Current
%
B
(a) (b)
RFP AA
NaCl
D-Glucose
Creatanine UA
Urea
Glycine
L-cysteine
0
20
40
60
80
100
120
A
Change
in
Current
%
E
H2
O2
Figure: (a) Interference study of Mod. GCE for the detection of RFP in presence of various biological compound (ratio 1:10) and
(b) Stability assessment in presence of RFP in 0.1 M PBS (pH = 7.4).
Interference and Reproducibility Test

20. 20
0.2 0.4 0.6 0.8 1.0
-15
-10
-5
0
5
10
15
Current
(mA)
Potential (V) vs. Ag/AgCl
(i)
(ii)
(iii)
(iv)
(a) (b)
5 10 15 20 25 30
0
5
10
15
20
25
30
-Z"
/
W
(
´10
2
)
Z' / W (´102
)
6 8 10 12
0
1
2
3
4
-Z"
/
W
(´10
2
)
Z' / W (´102
)
(a) CV of (i) bare, (ii) GO
modified, (iii) CuO modified and
(iv) GO@CuO/GCE in 1 mM
K3[Fe(CN)6] prepared in 0.1 M
KCl solution (b) Nyquist plot of
(i) & (ii) bare GCE in absence
and presence of RFP, (iii) & (iv)
GO@CuO/GCE in absence and
presence of 15 μM RFP in 1 mM
K3[Fe(CN)6] in 0.1 M KCl
0.2 0.4 0.6 0.8 1.0
-15
-10
-5
0
Current
Potential (V) vs. Ag/AgCl
(i)
(ii)
(iii)
(iv)
Parameter Bare GCE Bare GCE + RFP CuO@rGO/GCE CuO@rGO/GCE +
RFP
Rs (Ohm) 8.78 9.1 8.9 9.5
CPE (γ0)
(S-sec.n )
8.13×10-7 6.618×10-7 0.0009567 0.001182
Freq. power,
n
0.8 0.8 0.6866 0.8
R1 (Ohm) 667.7 600.3 560.3 538.4
Warburg (γ0)
(S-sec.5)
0.001684 0.001747 0.001627 0.001846
C (F) 1.32×10-7 3.319×10-7 0.02847 0.02639
R2 (Ohm) 198.4 496 158 251.6
χ2 2.546×10-5 1.367×10-4 1.896 ×10-5 7.803×10-5

21. 21
525 530 535 540 545
Metal-O
-OH
Raw data
Metal-O
-OH
C-O
C=O
Convoluted
Intensity
(arb.
unit)
Binding Energy (eV)
C=O
C-O
(a) (b)
(c) (d)
930 940 950 960
Satellite peaks
2p1/2
2p1/2
Satellite peaks
Convoluted
Raw data
2p3/2
2p3/2
Satellite peaks
Satellite peaks
Intensity
(arb.
unit)
Binding Energy (eV)
2p3/2
2p1/2
CuO Satellite
peaks CuO Satellite
peaks
282 285 288 291 294
O-C=O
Raw Data
C-O
C-C
C-OH
C=O
O-C=O
Convoluted
C=O
C-OH
C-C
C-O
Intensity
(arb.
unit)
Binding Energy (eV)
Model Lorentz
Equati y = y0 + (2*A/pi)*(w/(4*(x-xc)^2 + w^2))
Plot Peak1(Subt Peak2(SubtrPeak3(SubtrPeak4(Subtr
y0 -1095.3796 -1095.3796 -1095.3796 -1095.3796
xc 284.59641 284.95695 285.4176 ± 286.39561
w 0.57136 ± 0 0.61782 ± 0 0.86422 ± 0 1.46857 ± 0
A 60710.8365 80844.4283 61891.5659 61376.7590
Reduc 3624903.13424
R-Squ 0.99595
Adj. R- 0.99561
0 200 400 600 800 1000 1200
Intensity
(arb.
unit)
Binding Energy (eV)
XPS wide
C Cu
O

22. 22
4 5 6 7 8
0.40
0.45
0.50
0.55
0.60
0.65
Potential,
V
vs.
Ag/AgCl
pH
y = -0.053pH + 0.82
R2
= 0.99
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
-15
-10
-5
0
5
Current
(mA)
Potential, V vs. Ag/AgCl
8.0
7.4
6.5
5.5
4.5
4.0
(a) (b)
4 5 6 7 8
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Current
(mA)
pH
(c)

23. 23
-2 e-
Oxidation
+2 e-
Reduction
Rifampicin
(Hydroquinone form)
Rifampicin
(Quinone form)
-2 e-
Oxidation
+2 e-
Reduction
Rifampicin
(Hydroquinone form)
Rifampicin
(Quinone form)